User:J. C. Martinez-Garcia/Notebook/HMS Activities/2008/12/18

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These last weeks

These last weeks I have been involved in several activities, and it is time now to make a summary of them.

Protein purification.

These last weeks I have been involved in protein purification techniques. The procedure can be described in chronological terms as follows.

  1. Cultivate the Pichia Pastoris organisms. This organisms is first modified through recombinant genetic engineering to express a designed protein. In thsi case pichia is chosen because does not secrete protein, and then the modification makes it to secrete the designed protein and then makes easy to recover it. The modified protein is essentially a modified version of Erythropoietin. Pichia pastoris is a methylotrophic yeast. The expresion process is based on the work performed by two genes which code alcohol oxidase -AOX1 and AOX2-. Pichia uses methanol as its only source of carbo, and the first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular ooxygen by the enzyme alcohol oxidase. The promoter regulating the production of alcohol oxidase is the one used to drive heterogeneous protein expression in Pichia. The expression of the AOX1 gene is tighhtly regulated and induced by methanol to very high levels (the AOX1 gene has been isolated and a plasmid-borne version of the AOX1 promoter is used to drive expression of the gene of interest encoding the desired heterologous protein). AOX2 makes growth in methanol much slower than with AOX1 and this allows isolation of MutS strains. The wild strain is called Mut+, which is the one that metabolizes methanol. The pichia strain used in this case was modified to express the protein through secretion. The EPO version expressed by EPO is a glycoprotein. We used two different strains: 144 and 149, which produces proteins only different by an aminoacid. The vector used to utilize the pAOX1 promoter was pPICα. The protein production started cultivating chosen colonies of pichias originated from a single organism, once the good strain was identified.
  2. Concentration. Once the protein was secreted it was necessary to concentrate the protein. These procedure required the application of Centricon Plus 70 devices (special filters which allows the proteins contained in the solution -just produced by pichia, and once thsi one has been eliminated through centrifugation- to be separated by centrifugation).
  3. Purification. The purification process was performed via centrifugation and the application the probond resin by invitrogen under native conditions (i.e. the biological activity of the heterologous protein is not eliminated duyring the purification process). The resin recovers from the concentrated cellular products the protein required. The resin works works via binding to the His6 tag of the secreted protein.
  4. Filtering of the Imidazole. Once the protein has been purified it is necessary to eliminate the Imidazole and this done via the application of Bio-Rad’s Poly-Prep Chromatography Columns.
  5. Final concentration. Once the imidazole was eliminated from the protein (using PBS as buffer) the final purification process was performed. This procedure used again centrifugation (using a special array consisting in a two container pipes, one with a filter; icon concentrators 7mL/9K). The final processes finished when the protein was concentrated to a volumen of more or less 500μL.
  6. Measuring the concentration. The concentration of the final sample was performed using a spectrophotometer by nanodrop ND 1000.

The must tricky questions in all this procedure are the following ones:

  • The production of the medium to nurrish the pichias must be done in a very careful way. In my case some organism invaded my product and it was then necessary to produce it again.
  • The preparation of the gel must be very careful if it is not convenient or too much expensive to use a gel available in the market.
  • All the material must be correctly marked to avoid any kind of possible confusions.
  • The purification process must be done in the cool room (4°C).
  • Denaturated samples must be stocked at -20°C to avoid degradation.
  • When stocking protein or biological products at -80°C it is necessary to be completely sure that no liquid remaisn in not frozen after the corresponding sample was sank in liquid nitrogen.

Just to finish this text, once the protein were purified, a comassie was run to see if everything was OK. The result is captured by the following image, which display the distained gel:

Protein final.png